Confluent HFF monolayers grown on coverslips in 24-well plates were infected with freshly lysed parasites. Infected host cells were fixed with 4% paraformaldehyde in PBS, blocked for 30 min in 3% BSA+PBS, and permeabilized with 0.2% Triton X-100 (PBS-T) for 10 min. Primary antibodies (see below) diluted in 3% BSA-PBS were applied overnight at 4°C. Cultures were washed three times for 10 min each with PBS. Secondary antibodies diluted in 3% BSA-PBS were applied for 1hr at room temperature. Coverslips were washed three times for 10 min and 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI, Life Technologies; #D1306) diluted 1:1,000 in PBS was applied for 10 mins at room temperature. Following three 10 min washes, coverslips were mounted using Vectashield antifade mounting medium (Vector Labs; #H-1000).
The following primary antibodies were used at the dilutions indicated: rat anti-HA (1:2,000; Roche #11867423001), rabbit anti-HA (1:2,000; Cell Signaling Technology #3724), mouse anti-TgF1B-ATPase (1:4,000; 53 (link),72 (link)), rat anti-TgSORTLR (1:2,000; 52 (link)) and mouse anti-TgSERCA (1:2,000; 51 (link)). Secondary antibodies conjugated to a fluorophore (Alexa Fluor; Thermo Fisher #A11005, #A11006, #A11007, #A11034) were used for IFAs at a 1:5,000 dilution.
The following primary antibodies were used at the dilutions indicated: rat anti-HA (1:2,000; Roche #11867423001), rabbit anti-HA (1:2,000; Cell Signaling Technology #3724), mouse anti-TgF1B-ATPase (1:4,000; 53 (link),72 (link)), rat anti-TgSORTLR (1:2,000; 52 (link)) and mouse anti-TgSERCA (1:2,000; 51 (link)). Secondary antibodies conjugated to a fluorophore (Alexa Fluor; Thermo Fisher #A11005, #A11006, #A11007, #A11034) were used for IFAs at a 1:5,000 dilution.
